Bacillus thuringiensis toxin receptors and uses thereof

ABSTRACT

The invention relates to identification and characterization of recombinant DNA and polypeptides for specific Bt toxin receptors. In particular, the Bt toxin receptors of the invention include those derived from the Lepidopteran super family including the species  Trichoplusiani ni, Pseudoplusia includens, Helicoverpa zea , and  Spodoptera frugiperda . The receptors of the invention further include those derived from the Coleopteran super family and particularly from the species  Diabrotica virgifera virgifera . The recombinant DNA and polypeptides so provided are useful in the identification and design of novel Bt toxin receptor ligands including novel or improved insecticidal toxins for use in a variety of agricultural applications. Materials and methods for identifying novel toxins are also disclosed herein. The invention also provides methods for selecting toxins to combine to control insect populations by manipulating Bt toxin receptor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Provisional Appl. Ser. No.61/907,492, filed Nov. 22, 2013, the entire disclosure of which isincorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

The sequence listing contained in the file named “59644_a_ST25.txt”,which is 1,015,808 bytes (measured in operating system MS-Windows) andwas created on Nov. 14, 2014, is contemporaneously filed by electronicsubmission (using the United States Patent Office EFS-Web filing system)and incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The field of the invention relates to isolation and characterization ofrecombinant nucleic acid and polypeptides for Bacillus thuringiensis(Bt) toxin receptors. This invention further relates to methods ofidentifying and designing toxin receptor ligands including novel orimproved insecticidal toxins as well as the development of enhancedassays and assay methods, including array diagnostics and kits fordetermining receptor ligand interactions and effectiveness of certaininsecticidal polypeptides.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis (Bt) is a spore-forming Gram-positive bacterium.During sporulation, Bt produces proteinaceous inclusions which arecomposed of proteins known as Cry proteins. With their relatively highspecificity for particular insect pests and their general level ofsafety for man and the environment, Cry proteins have been used asbiopesticides for decades. Bt strains are classified into subspecies orvarieties, based on biochemical and serological criteria (de Barjac,ENTOMOPHAGA 7: 5-61 (1962); de Barjac). Certain Cry toxins derived fromBt are insecticidal and may be used for insect control. Their primaryaction is to lyse midgut epithelial cells in susceptible insect species.Cry toxins are first ingested as protoxins which are then solubilizedand proteolytically converted to smaller, protease-stable polypeptides,in the insect midgut. These activated toxins, also called toxic core,then bind to specific receptors at the surface of midgut epithelialcells, allowing them to insert into the membrane and form pores whichare permeable to small molecules such as inorganic ions, amino acids andsugars causing extensive damage and disruption to insect cells.Destruction of the cells results in extensive damage to the midgutepithelial tissue and death of the insect.

Specific binding of endotoxin to specific receptors located in theinsect midgut is one step in the mode of insecticidal action. Cry toxinsinteract sequentially with multiple receptors (Gómez et al. (2007)PEPTIDES, 28(1):169-7; Vachon et al. J (2012) INVERTEBR. PATHOL.,111(1):1-12.). For Cry1A toxins (Lepidopteran specific toxins), at leastfive different protein receptors have been described to be involved inthe cascade of interactions: a cadherin-like protein (“CADR”), aglycosylphosphatidyl-inositol (GPI)-anchored aminopeptidase-N (APN), aGPI-anchored alkaline phosphatase (ALP) and a 270 kDa glycoconjugatetransmembrane ABC transporter. Recently, it has been reported that an “ADisintegrin And Metalloprotease” or “ADAM” metalloprotease is a Cry3Aatoxin Coleopteran receptor (Ochoa-Campuzano et al. (2007) BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATION 362, 437-442). In addition, ithas been proposed that glycolipids are also important Cry-receptormolecules in insects and nematodes.

A threat to the use of Cry toxins is the development of insectresistance. No single glycoprotein appears to be essential for Cry1Atoxicity; e.g. variants of Cry1Ac which eliminate binding to a 115 kDaAPN only result in a two-fold decrease in toxicity (Raj agopal et al.(2002) J BIOL CHEM., 277:46849-51). RNA interference directed againstmidgut APNs produces a measurable but only slight decrease of Cry1Actoxicity. Therefore it has been suggested that the main significance ofCry1A toxin binding to these glycoproteins seems to be to an increase inthe concentration of the pre-pore oligomer at the membrane surface,acting to increase the probability of eventual insertion into themembrane of the pore forming portion of the toxin by some othermechanism.

One mechanism of resistance to Cry toxins is the interruption oftoxin-receptor interactions. Reduced levels of membrane-bound alkalinephosphatase are common to Lepidopteran strains resistant to Cry toxinsderived from Bacillus thuringiensis (Jurat-Fuentes et al. (2011) PLOSONE. 6(3):e17606. doi: 10.1371/journal.pone.0017606.). A map-basedcloning approach using a series of backcrosses identified ABC(ATP-binding cassette) transporter ABCC2 as the resistance gene in thecotton pest Heliothis virescens (Gahan et al. (2010) PLOS GENET.6(12):e1001248. doi: 10.1371/journal.pgen.1001248). An inactivatingmutation in this gene is genetically linked to Cry1Ac resistance and iscorrelated with loss of Cry1Ac binding to membrane vesicles.

Therefore, identification of Bt toxin receptors in insects and thereceptors' utility for changing or modulating resistance to various Bttoxins can be useful for investigating overall Bt toxin-Bt toxinreceptor interactions, selecting and designing improved toxins,developing novel pesticides and/or the creation of new Bt toxinresistance management strategies.

SUMMARY OF THE INVENTION

One aspect of the present invention provides recombinant receptorpolypeptides that are involved in Bt toxin binding, in which therecombinant receptor polypeptide has Bt toxin binding activity and hasan amino acid sequence selected from the group consisting of: a) SEQ IDNO: 23 through 44, 92 through 138, 143 through 146, and 168 through 186;b) an amino acid sequence having at least 80% sequence identity to theamino acid sequence set forth in SEQ ID NO: 23 through 44, 92 through138, 143 through 146, and 168 through 186; c) an amino acid sequencehaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at 99%sequence identity to an amino acid sequence set forth in SEQ ID NO: 23through 44, 92 through 138, 143 through 146, and 168 through 186; and e)an amino acid sequence consisting of the ligand binding region as setforth in SEQ ID NO: 143 through 146.

Another aspect of the present invention provides recombinant DNA thatencodes the receptor polypeptides or fragment thereof, which is orcomplementary to a sequence selected from the group consisting of SEQ IDNO: 1 through 22, 45 through 91, 139 through 142, and 149 through 167.

Another aspect of the present invention provides antibodies that bind tothe recombinant receptor polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO: 23 through 44, 92through 138, 143 through 146, and 168 through 186, or fragments thereof.

Another aspect of the present invention provides recombinant DNA vectorscomprising a nucleotide sequence encoding the recombinant receptorpolypeptide disclosed herein. The recombinant DNA vector can furthercomprise a promoter for expressing the recombinant receptor polypeptideeither in a prokaryotic or eukaryotic cell.

Yet another aspect of the present invention provides methods ofscreening for ligands that bind Bt toxin receptors, which methodscomprise the steps of: a) providing at least one Bt toxin receptorcomprising a recombinant receptor polypeptide disclosed herein; b)contacting the receptor polypeptide with a sample; and c) determiningbinding characteristics of the sample ligand.

Yet another aspect of the present invention provides methods to assessthe binding affinity of a candidate ligand for a receptor polypeptidedisclosed herein, which method comprises the steps of: a) contacting thecandidate ligand with the receptor; and b) measuring the bindingaffinity of the candidate ligand bound to the receptor.

Yet another aspect of the present invention provides methods to assessthe cytotoxicity of a candidate ligand, which method comprises the stepsof: a) contacting the candidate with cells that express the toxinreceptor comprising a receptor polypeptide disclosed herein; and then,b) measuring the cytotoxicity effect of the candidate ligand on thecells in terms of cell death indices.

Yet another aspect of the present invention provides a method to assessthe binding affinity of a first candidate ligand for an insect receptorcomprising a receptor polypeptide disclosed herein under the presence ofa second candidate ligand, comprising the steps of: a) contacting theinsect receptor with a first concentration of a first candidate ligand;b) measuring the binding affinity of the first candidate ligand; c)contacting the insect receptor with a second concentration of a secondcandidate ligand, d) measuring the binding affinity of the firstcandidate ligand, e) determining whether and how the presence of thesecond candidate ligand influences the binding affinity of the firstcandidate ligand; and optionally, f) repeating steps c) through e) withincreasing concentrations of the second candidate to determine candidateligands or combinations thereof of particular interest for use inagricultural applications including transgenic plants.

Yet another aspect of the present invention provides a method toengineer a candidate synthetic ligand containing domains or specifiedregions of ligands disclosed herein that—demonstrates an increasedbinding affinity for a specified insect receptor comprising a receptorpolypeptide disclosed herein or selected domains thereof linked to otherreceptor domains to comprise a complete ligand, which has the steps ofa) contacting the insect receptor with a first candidate ligand, b)measuring the binding affinity of the first candidate ligand, c)engineering the first candidate ligand to comprise a second candidateligand with variations in the domains selected that together comprisethe second candidate ligand, d) contacting the insect receptor with thesecond candidate ligand, and e) measuring the binding affinity of thesecond candidate ligand, and f) repeating steps c) through e) until acandidate ligand exhibits increased binding affinity for the insectreceptor of interest.

Yet another aspect of the present invention provides methods forselecting insect toxins to combine for controlling insect populations,which have the steps of a) reducing at least one receptor of the insecttoxin in a insect population, b) providing the insect population withthe reduced receptor at least one insect toxin, c) assessing toxicity ofthe toxin in the insect population, d) optionally repeating steps ii)and iii) to assess toxicity of additional toxins, and e) selecting onetoxin with reduced toxicity to combine with at least another toxin withunreduced toxicity.

One non-limiting embodiment of the present invention is using genesuppression to reduce receptor expression. Reducing the receptorexpression can be done by contacting an insect population with apolynucleotide comprising at least 18 contiguous nucleotides with asequence of about 95% to about 100% identity with a segment ofequivalent length of a DNA having a sequence selected from the groupconsisting of SEQ ID NO: 1 through 22, 45 through 91, 139 through 142,and 149 through 167, or the DNA complement thereof.

One non-limiting embodiment of the present invention is to combineCry3Bb and TIC1201 to control insect populations.

Another aspect of the present invention provides methods for selectinsect toxins to combine for controlling insect species including, butnot limited to, Trichoplusiani, Pseudoplusia includes, Helicoverpa zea,Spodoptera frugiperda, and Diabrotica virgifera virgifera.

Yet another aspect of the present invention provides transgenic hostcells co-expressing insect toxins selected by the methods providedherein. The transgenic host cells contemplated by the present inventioninclude, but not limited to, plant cell, bacterium, or plant seed.

Fertile transgenic plants expressing a Cry protein developed and/ordiscovered through the methods of the current invention may be testedfor insecticidal activity, and the plants showing optimal activityselected for further breeding. Methods are available in the art to assayfor insect activity. Generally, the protein is mixed and used in feedingassays. See, for example Marrone et al. (1985) J. OF ECONOMIC ENTOMOLOGY78:290-293. The present invention may be used for transformation of anyplant species, including, but not limited to, monocots and dicots.Examples of plants of interest include, but are not limited to, corn(maize), soybean, rape seed, cotton, alfalfa, sugar beet, rice, sugarcane, sorghum, wheat, tomato, crucifers, peppers, potato, tobacco,barley, rye, safflower, peanuts, sweet potato, cassava, coffee, coconut,pineapple, citrus trees, banana, avocado, fig, guava, mango, olive,papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, andconifers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that 1A. the partial protein structure of Cadherinreceptor including the Toxin binding region (TBR)2 and TBR3; The presentinvention designed the constructs to express receptor fragments based onthe partial structure. 1B. and 1C. the alignment of Cadherin receptorsfrom CBW=Cotton Bollworm; TBW=Tobacco Budworm; THW=Tobacco Hornworm;SW=Silkworm; FAW=Fall Armyworm; BAW=Beet Armyworm; CBL=Cabbage Looper;SBL=Soybean Looper; and the mutations in TBR2 and 3 respectively forTnCAD fragment made by the present invention.

FIG. 2 shows ligand blot analysis of Cry1Ab and Cry1Ac with TnCAD TBR2and TBR3 variants. The first four lanes contain alternating replicateMaltose Binding Protein (MBP)-fused or Tobacco Vein Mottled Virus (TVMV)cleaved TnCAD TBR2 variant protein expression extracts. The next fourlanes contain alternating replicate MBP-fused or TVMV cleaved TnCAD TBR3variant protein expression extracts. Negative control (−) contains Ecoli lysate without TnCAD and positive control (+) contains the trypticcore (TC) of the toxin used as the probe in the blot.

FIG. 3 shows that Cry1Ab binds to TnCAD TBR3 variant by a “pulldown”process, gel filtration and Biacore analyses. FIG. 3A shows gelfiltration analysis of the Cry1Ab and TnCAD TBR3 variant complex. Insetis the SDS-PAGE gel of representative fractions for the complex. FIG. 3Bshows NiNTA immobilized TnCAD TBR3 variant as bait for the tryptic coreof Cry1Ab. FIG. 3C shows Biacore binding traces for immobilized TnCADTBR3 variant with the listed toxins. CAD-TBR3 refers to the TBR3 variantform and CAD-WT refers to the corresponding wild type TnCAD fragment.

FIG. 4A illustrates the design of two truncation variants of TnCAD-TBR3variant A (SEQ ID NO: 143), and B (SEQ ID NO: 144). FIG. 4B shows thatgel filtration analysis demonstrating that TnCAD-TBR3 variant A withonly the membrane proximal domain (MPD) was co-eluted with Cry1Ab toxinin peak4. Peaks 1 and 2 are Cry1Ab and TnCad-TBR variant B alonerespectively as controls. FIG. 4C shows that gel filtration analysisdemonstrating that TnCAD-TBR3 variant B with only a truncated membraneproximal domain (MPD) was co-eluted with Cry1Ab toxin in peak2.

FIG. 5 illustrates that SPR experiments were performed using a BiacoreT000 instrument for SfALP and various toxins with BSA as the runningbuffer and control. FIG. 5A shows the binding characteristics of SfALPto Cry2AB. FIG. 5B shows the binding characteristics of ALP to Cry1Ca.FIG. 5C shows while Cry2AB and Cry1Ca bind to SfALP, Cry1AC and Tic105do not bind to SfALP.

FIG. 6 shows that PiCAD TBR3 variant resulted in cell sensitivity toCry1Ac and TIC107. FIG. 6A shows the effect of toxin challenge on SF9cells expressing the WT PiCAD full length coding sequence. FIG. 6B showsthe effect of toxin challenge on SF9 cells expressing the PiCAD TBR3variant. FIG. 6C shows the amino acid changes introduced into PiCAD TBR3variant provided by the present invention.

FIG. 7 shows that Spodoptera frugiperda ABC transporter is a functionalreceptor for Cry1A toxins. FIG. 7A shows that SF9 cells expressing ABCtransporter are sensitive to 50 ppm of tryptic cores for Cry1Ac1 andTIC107. Upper panels show the sytox green staining signal and lowerpanels show the bright field image of the corresponding region. FIG. 7Bshows the quantification of cell toxicity response as measured by asytox green signal.

FIG. 8 shows that suppressing either Cadherin or ADAM metalloprotease inWestern corn rootworm by dsRNA conferred Cry3Bb resistance measured byreduced mortality or 2nd/3rd-instar stunting on the tenth day whileTIC1201 remained effective, and that suppressing both Cadherin and ADAMmetalloprotease simultaneously had a synergistic effect in conferringCry3Bb resistance.

BRIEF DESCRIPTION OF THE SEQUENCES

NUC SEQ ID in Table 1 is the sequence number of the recombinant DNA inthe sequence listing

PEP SEQ ID in Table 1 is the sequence number of the recombinantpolypeptide in the sequence listing

TABLE 1 NUC PEP SEQ SEQ Gene Identifier ID ID AnnotationTrichoplusia_ni_ALP1 1 23 Alkaline phosphatase Trichoplusia_ni_APN1 2 24Aminopeptidase Trichoplusia_ni_APN6 3 25 AminopeptidaseTrichoplusia_ni_Cadherin_D01 4 26 cadherin like proteinTrichoplusia_ni_Cadherin_E02 5 27 cadherin like proteinTrichoplusia_ni_Cadherin_ 6 28 cadherin variant TBR3_CR9-TMDPseudoplusia_includens_APN1 7 29 Aminopeptidase Pseudoplusia_includens_8 30 cadherin like Cadherin_clone A01 protein Pseudoplusia_includens_ 931 cadherin like Cadherin_clone C01 protein Pseudoplusia_includens_Cad-10 32 cadherin variant herin_clone C01-TBR3variant Helicoverpa_zea_APN111 33 Aminopeptidase Helicoverpa_zea_ALP1 12 34 Alkaline phosphataseHelicoverpa_zea_ALP2 13 35 Alkaline phosphatase Helicoverpa_zea_APN3 1436 Aminopeptidase Spodoptera_frugiperda_ABC_ 15 37 ABC transportertransporter Spodoptera_frugiperda_Alkaline_ 16 38 Alkaline phosphatase_1phosphatase Spodoptera_frugiperda_ 17 39 AminopeptidaseAminopeptidase_N1 Diabrotica_virgifera_virgifera_ 18 40 ADAMADAM_metalloprotease_v1 metalloprotease Diabrotica_virgifera_virgifera_19 41 ADAM ADAM_metalloprotease _v2 metalloproteaseDiabrotica_virgifera_virgifera_ 20 42 ADAM ADAM_metalloprotease_Vnmetalloprotease Diabrotica_virgifera_virgifera_ 21 43 ABCABC_transporter_Vn transporter Diabrotica_virgifera_virgifera_ 22 44Aminopeptidase APN2 Diabrotica_virgifera_virgifera_ 45 92 ABCABC_transporter_105_10 transporter Diabrotica_virgifera_virgifera_ 46 93ABC ABC_transporter_105_9 transporter Diabrotica_virgifera_virgifera_ 4794 ABC ABC_transporter_218_1 transporter Diabrotica_virgifera_virgifera_48 95 ABC ABC_transporter_218_2 transporterDiabrotica_virgifera_virgifera_ 49 96 ABC ABC_transporter_218_3transporter Diabrotica_virgifera_virgifera_ 50 97 ABCABC_transporter_218_4 transporter Diabrotica_virgifera_virgifera_ 51 98Aminopeptidase Aminopeptidase_837_1 Diabrotica_virgifera_virgifera_ 5299 Aminopeptidase Aminopeptidase_837_2 Diabrotica_virgifera_virgifera_53 100 Aminopeptidase Aminopeptidase_871_1Diabrotica_virgifera_virgifera_ 54 101 cadherin like cadherin_1083_1protein Diabrotica_virgifera_virgifera_ 55 102 cadherin likecadherin_1817_1 protein Diabrotica_virgifera_virgifera_ 56 103 ABCABC_transporter_01859_1 transporter Diabrotica_virgifera_virgifera_ 57104 ABC ABC_transporter_01867_1 transporterDiabrotica_virgifera_virgifera_ 58 105 ABC ABC_transporter_01873_1transporter Diabrotica_virgifera_virgifera_ 59 106 AminopeptidaseAminopeptidase_01949_1 Diabrotica_virgifera_virgifera_ 60 107 ADAMADAM_metalloprotease_01952_1 metalloproteaseDiabrotica_virgifera_virgifera_ 61 108 AminopeptidaseAminopeptidase_02024_1 Diabrotica_virgifera_virgifera_ 62 109Aminopeptidase Aminopeptidase_02031_1 Diabrotica_virgifera_virgifera_ 63110 Aminopeptidase Aminopeptidase_02119_1Diabrotica_virgifera_virgifera_ 64 111 ADAM ADAM_metalloprotease_02122_1metalloprotease Diabrotica_virgifera_virgifera_ 65 112 AminopeptidaseAminopeptidase_02140_1 DIADiabrotica_virgifera_ 66 113 Aminopeptidasevirgifera_Aminopeptidase_02340_1 Diabrotica_virgifera_virgifera_ 67 114ABC ABC_transporter_02470_1 transporter Diabrotica_virgifera_virgifera_68 115 ABC ABC_transporter_02630_1 transporterDiabrotica_virgifera_virgifera_ 69 116 Alkaline ALP_02713_1 phosphataseDiabrotica_virgifera_virgifera_ 70 117 AminopeptidaseAminopeptidase_03898_1 Diabrotica_virgifera_virgifera_ 71 118 ADAMADAM_metalloprotease_04620_1 metalloproteaseDiabrotica_virgifera_virgifera_ 72 119 ADAM ADAM_metalloprotease_04627_1metalloprotease Diabrotica_virgifera_virgifera_ 73 120 AminopeptidaseAminopeptidase_04697_1 Diabrotica_virgifera_virgifera_ 74 121Aminopeptidase Aminopeptidase_04881_1 Diabrotica_virgifera_virgifera_ 75122 cadherin cadherin_04907_1 Diabrotica_virgifera_virgifera_ 76 123Aminopeptidase Aminopeptidase_05042_1 Diabrotica_virgifera_virgifera_ 77124 ADAM ADAM_metalloprotease_05390_1 metalloproteaseDiabrotica_virgifera_virgifera_ 78 125 ABC ABC_transporter_05581_1transporter Diabrotica_virgifera_virgifera_ 79 126 ADAMADAM_metalloprotease_05844_1 metalloproteaseDiabrotica_virgifera_virgifera_ 80 127 ABC ABC_transporter_06637_1transporter Diabrotica_virgifera_virgifera_ 81 128 ADAMADAM_metalloprotease_07383_1 metalloproteaseDiabrotica_virgifera_virgifera_ 82 129 ABC ABC_transporter_07661_1transporter Diabrotica_virgifera_virgifera_ 83 130 ADAMADAM_metalloprotease_08650_1 metalloproteaseDiabrotica_virgifera_virgifera_ 84 131 AminopeptidaseAminopeptidase_08778_1 Diabrotica_virgifera_virgifera_ 85 132Aminopeptidase Aminopeptidase_08810_1 Diabrotica_virgifera_virgifera_ 86133 Aminopeptidase Aminopeptidase_09768_1Diabrotica_virgifera_virgifera_ 87 134 cadherin like cadherin_10167_1protein Diabrotica_virgifera_virgifera_ 88 135 ADAMADAM_metalloprotease_10594_1 metalloproteaseDiabrotica_virgifera_virgifera_ 89 136 ABC ABC_transporter_11225_12?transporter Diabrotica_virgifera_virgifera_ 90 137 ABCABC_transporter_11255_1 transporter Diabrotica_virgifera_virgifera_ 91138 cadherin like cadherin_01803_1 protein Tn CAD variant A 139 143Cadherin variant TBR Tn CAD variant B 140 144 Cadherin variant TBR CRWABC transporter TBR 141 145 ABC transporter TBR APN2 TBR frag 9 142 146Aminopeptidase TBR dsRNA for cadherin 147 / / dsRNA for ADAMmetalloprotease 148 / / DsABCc2 149 168 ABC transporter DsABCc3 150 169ABC transporter DvABCa3 151 170 ABC transporter DvABCc1 152 171 ABCtransporter HzABCa7 153 172 ABC transporter HzABCb1 154 173 ABCtransporter PiABCb1 155 174 ABC transporter PiABCc2 156 175 ABCtransporter PiABCc3 157 176 ABC transporter SfABCa3 158 177 ABCtransporter SfABCb1 159 178 ABC transporter SfABCb5 160 179 ABCtransporter SfABCc1 161 180 ABC transporter SfABCc2 162 181 ABCtransporter SfABCc4 163 182 ABC transporter SfABCc5 164 183 ABCtransporter SfABCg2 165 184 ABC transporter HzABCc2 166 185 ABCtransporter HzABCc3 167 186 ABC transporter

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are receptor polypeptides isolated from various insectsthat are involved in Bt toxin binding including those derived from theLepidopteran superfamily, e.g. from the species Trichoplusia ni(Tn),Pseudoplusia includes (Pi), Helicoverpa zea, and Spodoptera frugiperda(Sf), and those derived from the Coleopteran superfamily, e.g. from thespecies Diabrotica virgifera virgifera. These receptor polypeptides havehomology to sequences present in cadherin, ABC transporter, Alkalinephosphatase, ADAM metalloprotease and/or Aminopeptidase sequences. Inparticular, provided herein are recombinant polypeptides comprising anamino acid sequence as set for in SEQ ID NOs: 23 through 44, 92 through138, 143 through 146, and 168 through 186 listed in Table 1, or,fragments or fusions thereof in which non-essential, or not relevant,amino acid residues have been added, replaced, or deleted. Furtherprovided are recombinant DNA comprising a nucleotide sequence as setforth in SEQ ID NOs: 1 through 22, 45 through 91, 139 through 142, and149 through 167 listed in Table 1.

In the context of the present invention, a homologous sequence is takento include an amino acid sequence which is at least 60, 70, 80 or 90%identical, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or99.5% identical, or identical to any fraction percentage in this rangeat the amino acid level over at least 20, 50, 100, 200, 300 or 400 aminoacids with the amino acid sequences set forth in SEQ ID NOs: 23 through44, 92 through 138, 143 through 146, and 168 through 186. In particular,homology should typically be considered with respect to those regions ofthe sequence known to be essential for the function of the protein suchas the ligand binding region as set forth in SEQ ID NO:137 through SEQID NO: 140. Recombinant polypeptides of the present invention alsocomprise a contiguous sequence having greater than 60, 70, 80 or 90%homology, or greater than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or99.5% homology, to one or more of amino acids of SEQ ID NOs: 23 through44, 92 through 138, 143 through 146, and 168 through 186.

Disclosed herein, the term “recombinant” indicates that the material(e.g., a cell, a nucleic acid, polypeptide or a protein) has beenartificially or synthetically (non-naturally) altered by humanintervention. The alteration can be performed on the material within orremoved from, its natural environment or state. For example, a“recombinant DNA” is one that is made by recombining nucleic acids,e.g., during cloning, DNA shuffling or other procedures; a “recombinantpolypeptide” or “recombinant protein” may be a polypeptide or proteinwhich is produced by expression of a recombinant nucleic acid.

The recombinant DNA and polypeptide disclosed herein encompass proteinvariants, or fragments thereof. In one embodiment, protein variantsinclude any amino acid polymers in which one or more amino acid residueis an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to any naturally occurring amino acidpolymers. The nature of such analogues of naturally occurring aminoacids is that, when incorporated into a protein that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. In anotherembodiment of the invention, protein variants are generated by deletionsand insertions. A protein “fragment” is a peptide or polypeptidemolecule whose amino acid sequence comprises a subset of the amino acidsequence of that protein. Specifically a fragment of a Bt toxin receptorrefers to the biologically active portion of a Bt toxin receptorpolypeptide. In another embodiment, these protein variants and fragmentscontinue to possess the desired toxin binding activity.

It is known in the art that proteins or polypeptides may undergoposttranslational modification, including but not limited to, disulfidebond formation, gamma-carboxylation of glutamic acid residues,glycosylation, lipid attachment, phosphorylation, oligomerization,hydroxylation and ADP-ribosylation. Modifications can occur anywhere ina polypeptide, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini. Blockage of the amino orcarboxyl group in a polypeptide, or both, by a covalent modification, isknown in naturally occurring and synthetic polypeptides and suchmodifications can be present in polypeptides of the present invention,as well. For instance, the amino terminal residue of polypeptides madein E. coli or other cells, prior to proteolytic processing, almostinvariably will be N-formylmethionine. During post-translationalmodification of the polypeptide, a methionine residue at the NH2terminus can be deleted. Accordingly, contemplated is the use of boththe methionine-containing and the methionine-less amino terminalvariants of the protein disclosed herein.

In one embodiment, provided herein is the use of structural informationfor the design and production of variant receptors that have alteredbinding properties and/or specificities to known toxins. In a specificembodiment, the present invention provides the variant receptors, forexample, as set for in SEQ ID NO: 28 and 32. The variants or modifiedforms of receptors disclosed herein may be prepared in a number of ways.For example, the wild-type receptor sequence can be mutated in thosesites identified using the present invention as desirable for mutation,by means of site directed mutagenesis by PCR, oligonucleotide-directedmutagenesis or other conventional methods well known to the personskilled in the art. Amino acid substitutions, deletions and/orinsertions can readily be made using peptide synthetic techniques wellknown in the art, such as solid phase peptide synthesis and the like, orby recombinant DNA manipulation. Methods for the manipulation of DNAsequences to produce substitution, insertion or deletion variants of aprotein are known in the art. For example, techniques for makingsubstitution mutations at predetermined sites in DNA are well known tothose skilled in the art and include M13 mutagenesis, T7-Gen in vitromutagenesis (USB, Cleveland, Ohio), QuickChange Site Directedmutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directedmutagenesis or other site-directed mutagenesis protocols.

Disclosed herein, the term “domain” refers to a set of amino acidsconserved at specific positions along an alignment of sequences ofevolutionarily related proteins. Specialist databases exist for theidentification of domains, for example, SMART (Schultz et al. (1998)PROC. NATL. ACAD. SCI. USA 95, 5857-5864; Letunic et al. (2002) NucleicAcids Res 30, 242-244), InterPro (Mulder et al. (2003) NUCL. ACIDS. RES.31, 315-318), Prosite (Bucher and Bairoch (1994), or Pfam (Bateman etal. (2002) NUCLEIC ACIDS RESEARCH 30(1): 276-280). A set of tools for insilico analysis of protein sequences is available on the ExPASyproteomics server (Swiss Institute of Bioinformatics (Gasteiger et al.(2003) ExPASy: the proteomics server for in-depth protein knowledge andanalysis, NUCLEIC ACIDS RES. 31:3784-3788). Domains or motifs can alsobe identified using techniques known in the art, such as by sequencealignment. While amino acids at other positions can vary betweenhomologues, amino acids that are highly conserved at specific positionsindicate amino acids that are likely essential in the structure,stability or function of a protein. Identified by their high degree ofconservation in aligned sequences of a family of protein homologues,they can be used as identifiers to determine if any polypeptide inquestion belongs to a previously identified polypeptide family.

A polypeptide or fragment thereof that comprises one or more additionalpeptide regions not derived from that protein is a “fusion” protein.Such molecules can be derivatized to contain carbohydrate or othermoieties (such as keyhole limpet hemocyanin, etc.). Fusion proteins orpeptide molecules of the present invention can be produced viarecombinant means.

In another embodiment, one or more of the polypeptide or fragment ofpeptide molecules can be produced via chemical synthesis, or byexpressing in a suitable prokaryotic or eukaryotic host. Methods forexpression are described by Sambrook, et al., (In: MOLECULAR CLONING, ALABORATORY MANUAL, 2nd Edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989)).

Another aspect of the present invention relates to antibodies,single-chain antigen binding molecules, or other proteins thatspecifically bind to one or more of the protein or recombinantpolypeptide disclosed herein and their homologues, fusions or fragments.Such antibodies can be used to quantitatively or qualitatively detectthe protein or peptide molecules of the present invention. As usedherein, an antibody or peptide is said to “specifically bind” to aprotein or peptide molecule of the present invention if such binding isnot competitively inhibited by the presence of non-related molecules. Inan embodiment, the antibodies bind to proteins disclosed herein.

Nucleic acid molecules that encode all or part of the recombinantpolypeptide disclosed herein can be expressed, via recombinant means, toyield protein or peptides that can in turn be used to elicit antibodiesthat are capable of binding the expressed protein or peptide. Suchantibodies can be used in immunoassays for that protein. Suchprotein-encoding molecules, or their fragments may be a “fusion”molecule (e.g., a part of a larger nucleic acid molecule) such that,upon expression, a fusion protein is produced. It is understood that anyof the nucleic acid molecules disclosed herein can be expressed, viarecombinant means, to yield proteins or polypeptides encoded by thesenucleic acid molecules.

The antibodies that specifically bind proteins and protein fragments ofthe present invention can be polyclonal or monoclonal, and can compriseintact immunoglobulins, or antigen binding portions of immunoglobulins(such as (F(ab′), F(ab′)2) fragments, or single-chain immunoglobulinsproducible, for example, via recombinant means). It is understood thatpractitioners are familiar with the standard resource materials whichdescribe specific conditions and procedures for the construction,manipulation and isolation of antibodies (see, for example, Harlow andLane, In Antibodies: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1988)).

In an embodiment, such antibody molecules or their fragments can be usedfor diagnostic purposes. Where the antibodies are intended fordiagnostic purposes, it may be desirable to derivatize them, for examplewith a ligand group (such as biotin) or a detectable marker group (suchas a fluorescent group, a radioisotope or an enzyme).

The ability to produce antibodies that bind the protein or polypeptidemolecules of the present invention permits the identification of mimeticcompounds of those molecules. A “mimetic compound” is a compound that isnot that compound, or a fragment of that compound, but which nonethelessexhibits an ability to specifically bind to antibodies directed againstthat compound.

In an embodiment, disclosed herein are recombinant DNA vectors inprokaryotic or eukaryotic hosts or cells. The recombinant DNA vectorscontemplated herein include those for cloning, expression andtransformation vectors. The recombinant DNA vector prepared forintroduction into a prokaryotic or eukaryotic host typically comprise areplication system (e.g. vector) recognized by the host, including theDNA fragment encoding the recombinant polypeptide disclosed herein, andwill preferably also include transcription and translational initiationregulatory sequences operably linked to the polypeptide-encodingsegment. A non-limiting example for expression systems (expressionvectors) can include an origin of replication or autonomouslyreplicating sequence (ARS) and expression control sequences, a promoter,an enhancer and necessary processing information sites, such asribosome-binding sites, RNA splice sites, polyadenylation sites,transcriptional terminator sequences, and mRNA stabilizing sequences.Signal peptides can also be included where appropriate from secretedpolypeptides of the same or related species, which allow the protein tocross and/or lodge in cell membranes or be secreted from the cell.

In another embodiment, expression and transformation vectors can containa selectable marker, that is, a gene encoding a protein necessary forthe survival or growth of a host cell transformed with the vector.Although such a marker gene may be carried on another polynucleotidesequence co-introduced into the host cell, it is most often contained onthe cloning vector. Only those host cells into which the marker gene hasbeen introduced survive and/or grow under selective conditions.Typically selection genes encode proteins that (a) confer resistance toantibiotics or other toxic substances, e.g., ampicillin, neomycin,methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c)supply critical nutrients not available from complex media. The choiceof the selectable marker depends on the host cell; appropriate markersfor different hosts are known in the art.

The term “operably linked”, as used herein, refers to a functionallinkage between at least two expression regulatory elements, such as,but not limited to the functional linkage between promoter sequence andgene of interest, such that the promoter sequence is able to initiatetranscription of the gene of interest. Another such non-limiting exampleis the functional linkage between signal peptide and gene of interest.

Another embodiment of the present invention relates to transgenic cellsor organisms transformed with recombinant DNA encoding Bt toxin receptordisclosed herein. The transgenic organisms or cells can be eitherprokaryotic or eukaryotic, for examples, insect, yeast, bacteria, phage,and fungus. The terms “transformation”, as used herein, alsoencompassing transfection, conjugation and transduction, include amultiplicity of publicly known methods for introducing recombinant DNAinto a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofectin,cellfectin, natural competence, chemically mediated transfer,electroporation or particle bombardment. Methods suitable fortransforming or transfecting host cells can be found in Sambrook et al.(MOLECULAR CLONING: A LABORATORY MANUAL., 2nd edition, Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989) and other laboratory manuals such as METHODS INMOLECULAR BIOLOGY, 1995, vol. 44, Agrobacterium protocols, eds: Gartlandand Davey, Humana Press, Totowa, N.J. Transformation encompassingtransfection, conjugation and transduction can be either transient orstable transformation. It is known about stable or transient integrationof recombinant DNA that, depending on the expression vector used andtransformation technique. For example, Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g. Sf9 cells) includethe pAc series (Smith et al. (1983) MOL. CELL BIOL. 3:2156) and the pVLseries (Lucklow et al. (1989) Virology 170:31), as well as commerciallyavailable derivatives.

Further provided herein are methods utilizing Bt toxin receptordisclosed herein to screen for candidate ligands for that receptor.Examples for such ligands include, but are not limited to, natural andmodified toxins, pesticides, antibodies, peptides, receptor agonists andantagonists, and other small molecules or domains (or segments) of knowntoxins designed or deduced to interact with the receptors disclosedherein. Candidate ligands include molecules available from diverselibraries of small molecules created by combinatorial synthetic methods.In addition to screening for candidate ligands, the screen can be usedto screen engineered toxins for improved forms containing domains (orsegments) from various sources which can be more specific or lessspecific to particular classes of insects as desired, or can be morepotent in killing a specific class of insects, or can be effective inkilling a class of insects with established resistance to Bt existingtoxins. The methods of engineering a Bt toxin include, but not limitedto, those protein design methods involving mutagenesis (Smith et al.(1994) BIOCHEM. J. 302:611-616 and Wu et al. (2000) FEBS Lett.473:227-232), deletion (Tabashnik et al. (2011) NATURE BIOTECHNOLOGY 29:1128-1131), addition, and domain substitution (Maagd et al. (1996) APPLENVIRON MICROBIOL. 62(5): 1537-1543). Furthermore, engineering toxinvariants and screening for improved forms can be carried out in a highthroughput manner.

In one embodiment, the method disclosed herein comprise providing a Bttoxin receptor in binding assays to determine differences between atleast two toxins or variants of the same toxin. Non-limiting examplesare the reconstitution of receptors in Brush Border Membrane Vesicles(BBMV) and their application in binding assays such as ligand blot,binding in solution, light scattering or Surface Plasmon Resonance, toobtain kinetic parameters, such as association/disassociation rates,binding affinity, binding site specificity and to obtain information ifBt toxin binding is reversible or irreversible.

In another embodiment, insect receptors are used for structure-functionanalysis. One such example is the identification of putative bindingregions in the toxin and receptor to design new toxin variants withstronger binding, broad spectrum of binding, or different specificity.

In yet another embodiment identified receptor sequences, or fragmentsthereof, are used as markers for identifying changes in allelefrequencies of different populations by determining different haplotypesand the frequency of appearance. As used herein “haplotype” is acombination of alleles at adjacent locations on a chromosome that areinherited together. A haplotype may be one locus, several loci, or anentire chromosome.

In one embodiment, the methods disclosed herein comprise providing atleast one Bt toxin receptor, contacting the Bt toxin receptor with asample containing a ligand candidate under conditions promoting binding,and determining the binding characteristics or the viability of the cellexpressing the Bt toxin receptor on cell surface.

As used herein, the term “conditions promoting binding” refers to anycombination of physical and biochemical conditions that enables a ligandto detectably bind the intended receptor polypeptide disclosed hereinover background levels. “Detectably binding” as used herein refers tosensing of receptor binding by various means, including, but not limitedto, loss of toxin function by feeding or injection of a target pest withone or more dsRNA targeting for suppression of a particular receptor orreceptor ligand. Examples of such conditions for binding of Cry1 toxinsto Bt toxin receptors, as well as methods for assessing the binding, areknown in the art and include, but are not limited to, those described inKeeton et al. (1998) APPL ENVIRON MICROBIOL 64(6): 2158-2165; Francis etal. (1997) INSECT BIOCHEM MOL BIOL 27(6):541-550; Keeton et al. (1997)APPL ENVIRON MICROBIOL 63(9):3419-3425; Vadlamudi et al. (1995) J BiolChem 270(10):5490-5494; Ihara et al. (1998) COMPARATIVE BIOCHEMISTRY ANDPHYSIOLOGY, PART B 120:197-204; and Nagamatsu et al. (1998) BIOSCI.BIOTECHNOL. BIOCHEM. 62(4):727-734.

In yet another embodiment, the screening assays can be whole organism,intact cell or in vitro assays which include exposing a ligand bindingregion or domain to a sample ligand and detecting the formation of aligand-receptor complex. A ligand binding region is the amino acidfragment of a receptor that binds a ligand. A ligand binding region canbe a fragment of a ligand binding domain. The assays could be directligand-receptor binding assays or ligand competition assays.

Methods are known for studying protein-protein interactions, such asyeast and/or bacterial two-hybrid systems (for example, CLONTECH (PaloAlto, Calif.) or Display Systems Biotech Inc. (Vista, Ca)), surfaceplasmon resonance (SPR, Richard B M Schasfoort and Anna J Tudos (2008).Handbook of Surface Plasmon Resonance), co-immunoprecipitation (PhizickyE. M. and Fields S. (1995) Protein-protein interactions: Methods fordetection and analysis. MICROBIOL REV. 59, 94-123.), pull-down assays(Einarson, M. B. (2001). Detection of Protein-Protein Interactions Usingthe GST Fusion Protein Pulldown Technique. IN MOLECULAR CLONING: ALABORATORY MANUAL, 3rd Edition, Cold Spring Harbor Laboratory Press, pp.18.55-18.59) and phage display (Sachdev S Sidhu et al. Exploringprotein-protein interactions with phage display. CHEMBIOCHEM. 2003 Jan.3; 4(1):14-25) and can be used for determining ligand-receptor binding.

For in-vitro binding assays, the polypeptide can be provided asisolated, lysed, or homogenized cellular preparations. Isolatedpolypeptides can be provided in solution, or immobilized to a matrix.Methods for immobilizing polypeptides are known in the art, and include,but are not limited to, construction and use of fusion polypeptides withcommercially available high affinity ligands. For example, GST fusionproteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtitre plates.The polypeptides can also be immobilized utilizing well techniques inthe art utilizing conjugation of biotin and streptavidin. Thepolypeptides can also be immobilized utilizing known techniques in theart utilizing chemical conjugation (linking) of polypeptides to amatrix. Alternatively, the polypeptides can be provided in intact cellbinding assays in which the polypeptides are generally expressed as cellsurface Bt toxin receptors.

In another embodiment, provided herein are methods utilizing intact celltoxicity assays to screen for ligands that bind to Bt toxin receptordescribed herein and confer toxicity upon a cell of interest expressingthe Bt toxin receptor. A ligand selected by this screening can be apotential insecticidal toxin to insects expressing the receptorpolypeptides, particularly enterally. The toxicity assays includeexposing, in intact cells expressing a receptor polypeptide of theinvention, the toxin binding region, domain or segment of thepolypeptide to a sample ligand and detecting the toxicity affected inthe cell expressing the receptor polypeptide. The term “toxicity” refersto the decreased viability of a cell. The term “viability” refers to theability of a cell to proliferate and/or differentiate and/or maintainits biological characteristics in a manner characteristic of that cellin the absence of a particular cytotoxic agent.

Yet in another embodiment, toxicity, binding and permeability can beanalyzed using BBMV prepared from insect midgut expressing the Bt toxinreceptor provided by the present invention. The BBMV preparation and itsuses in various assays are known in the art, for example as described inWolfersberger et al. (1987) COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A86, 301-308 and Luo et al. (1999) APPL ENVIRON MICROBIOL. 65(2):457-464. The insects used to prepare for BBMV can be transgenic insectsexpressing Bt toxin receptor provided by the present invention.Permeability derived from the BBMV based assay can be used as an indexfor the toxicity of a toxin for insect cells expressing the same Bttoxin receptor.

Yet in another embodiment, provided herein are methods to assess andcompare the binding affinities of at least two candidate ligands for aBt toxin receptor. In order to prevent or delay the onset of insectsdeveloping resistance against toxins and pesticides, new candidateligands having (or exhibiting) different Mode-of-Action, herein calledMOA, are needed. A different MOA can present itself in various ways; forexample novel candidate ligands can bind to different insect receptorsthan the toxins or pesticides currently in use. Discovery of novelinsecticidal toxins that bind to at least one different receptorcompared to another toxin, are amenable for use as insecticides thatprevent or delay the onset of insect resistance development. Severalmethods can be used to compare the MOA of one toxin to another. Onenon-limiting example includes the use of competition assays between thereceptor binding of a candidate ligand and a toxin. Alternatively, as anembodiment of the present invention, interfering a toxin and itsreceptor interaction by different methods, such as reducing receptorexpression by gene suppression in insects, can be used to differentiatetoxin MOA. RNAi methods for gene suppression in insects have beendescribed in Baum J A, et al. (2007) (Control of coleopteran insectpests through RNA interference. Nat Biotechnol 25: 1322-1326) andUS20090307803. Candidate ligands with different MOA can be stacked orcombined with other toxins in a transformation vector to bestowtransformed plants with multiple MOA resistance against given insectpests.

Several methods can be used to perform competition experiments for thebinding of at least one candidate ligand compared to at least one toxin.One example is binding assays with radio labeled candidate ligand ortoxin similar to the method described in Iracheta et al. (2005)(Screening for Bacillus thuringiensis Crystal Proteins Active againstthe Cabbage Looper, Trichoplusia ni, J. INVERTEBR. PATHOL., 76, 70-75)and Jimenez-Juarez et al. (2007) (Bacillus thuringiensis Cry1Ab MutantsAffecting Oligomer Formation Are Non-toxic to Manduca sexta Larvae, J.BIOL. CHEM. 282 (28), 21222-21229).

It should be understood that the entire disclosure of each referencecited herein is incorporated within the disclosure of this application.

The following examples are included to demonstrate aspects of theinvention. Those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificaspects which are disclosed and still obtain like or similar resultswithout departing from the spirit and scope of the disclosure.

Example 1. Identify Insect Receptors for Bt Toxin by Yeast Two HybridMethod

A yeast two-hybrid was performed to identify receptors for Cry3Bb toxin.The coding sequence for the Cry toxin was PCR-amplified and cloned intopB27 as a C-terminal fusion to LexA (N-LexA-Cry toxin-C). A cDNA librarywas created for midgut tissue collected from Diabrotica virgiferavirgifera for use as prey in the assay. For interaction analysis, theULTImate screen was performed by Hybrigenics (Paris, France). Results ofthis screen identified ABC transporter (SEQ ID NO: 21) as receptor forCry3Bb toxins.

Furthermore, yeast two-hybrid was also performed to identify fragmentsof either ABC transporter or Aminopeptidase that are sufficient to bindBt toxins.

TABLE 2 5′ terminal 3′ terminal Fragment nucleotide nucleotide 1 11661857 2 1166 1860 3 1166 1817 4 1166 1831 5 1196 1887 6 1196 1899 7 12631970 8 1383 1976 9 1461 2078 10 1461 2115 11 1130 1862 12 1128 1862 131242 1862 14 1127 1862 15 1321 1952 16 1311 1952 17 1487 2153 18 13831967 19 1481 2115 20 1494 2115

Interaction of ABC transporter fragments with Cry3Bb was demonstrated byyeast two-hybrid experiments. The 5′ and 3′ terminal nucleotides inTable 2 representing the start and end points of ABC transporterfragments are corresponding to the nucleotide position in the fulllength Diabrotica virgifera virgifera ABC transporter. Fragments 1-20 ofABC transporter were demonstrated to have positive interaction withCry3Bb.

TABLE 3 Aminopeptidase amino terminal carboxy terminal InteractionFragment amino acid amino acid with Cry3Aa 1 16 916 negative 2 16 174positive 3 16 249 positive 4 249 528 positive 5 505 916 negative 6 249916 negative 7 16 470 positive 8 16 470 positive 9 16 528 positive

Interaction of Aminopeptidase N (as set forth in SEQ ID NO: 22)fragments with Cry3Aa, TIC1201 or Cry3Bb was characterized by yeasttwo-hybrid experiments as shown in Table 3. Results with Cry3Bb andTIC1201 were all negative.

Example 2. Isolate Bt Receptor Genes

This example illustrates the isolation of Bt receptor genes from variousinsects exhibiting susceptibility to a particular Bt toxin.

All insects for these studies were obtained from the Monsanto insectory.RNA was isolated using the RNeasy Kit for high Lipid containing tissues(Qiagen, Valencia, Calif.). cDNA was transcribed using oligodT primersand Superscript III reverse transcriptase according to manufacturers'recommendations (Life Technologies, Carlsbad, Calif.). The geneinformation for ALP1, APN1, and APN6 from Trichoplusia ni was obtainedfrom NCBI locus identifiers AEG79734, AAX39863, and AAX39863respectively. Primers were designed based on this sequence for isolationof the full length transcript. For Helicoverpa zea ALP1, ALP2, and APN1,the sequences in the public database for Helicoverpa armigera were usedto design primers for isolation of the full length transcript. Thesequences for these genes were obtained from the following NCBI locusidentifiers: ALP1 from ACF40806, ALP2 from ACF40807, APN1 from AAQ57405and related sequences. Both ALP1 and ALP2 were cloned with primersdesigned to these sequences. However, APN1 from Helicoverpa armigera wassufficiently different at the C-terminus and thus 3′RACE was used todetermine the full coding region by extension of the transcript usingGeneRACER technology (Life Technologies, Carlsbad, Calif.). Anotheramplicon from the Helicoverpa zea 3′RACE reaction was APN3. This genewas also cloned and included in the analysis. Partial sequence forPseudoplusia includens Aminopeptidase N1 was obtained from Monsantoproprietary databases. The complete sequence was not available andtherefore 5′ and 3′ Rapid Amplification of cDNA Ends (RACE) wasperformed to isolate and confirm the complete coding region of thisgene. RACE was performed according to the GeneRacer kit (LifeTechnologies, San Diego, Calif.). Partial sequence for Diabroticavirgifera virgifera ADAM metalloprotease and ABC transporter wasobtained from Monsanto proprietary databases. RACE was performed toidentify the 5′ and 3′ coding sequences for ADAM and ABC transporter.Two variants for ADAM were identified where one possessed a predictedtransmembrane domain (Pfam) with a C-terminal extension and the othercontained only the predicted extracellular, N-terminal portion of thegene.

Based on the analysis of the amino acid sequence of SfALP1, the encodedsequence has an N-terminal secretion signal and a C-terminal GPI-anchorand transmembrane sequence, and one predicted N-linked glycosylationsite (Asn-261). SfALP1 is therefore a type-Ia transmembrane protein withthe bulk of the structure lying on the extracellular side of the plasmamembrane, and tethered at the C-terminus with a GPI-anchor.

Example 3. Engineering Cadherin (CAD) TBR2 and TBR3 Variants

This example illustrates the engineering of cadherin proteins to which atarget Bt toxin does not bind.

The sequence for Cadherin from Trichoplusia ni (hereafter referred to asTnCAD) was obtained from NCBI locus identifier AEA29692. Primers weredesigned based on this sequence for isolation of the full lengthtranscript. The first 1800 bp were amplified along with a secondamplification of the region from 1800 bp to the end of the gene to clonethe full length coding sequence for TnCAD. Overlapping PCR was used toextend the gene and clone it into a TOPO vector. Two versions of TnCADwere obtained.

The gene information for Pseudoplusia includens Cadherin (hereafterreferred to as PiCAD) was obtained, in part, from a Monsanto proprietarysequence collection for this organism by performing BLAST searches usingthe TnCAD sequence. Primers were designed to amplify and confirm 5′ and3′ ends of the transcript for PiCAD using RACE.

To clone the full length version of PiCAD, three regions of PiCAD wereindividually amplified and combined using overlapping PCR to obtain thecomplete coding region. The three regions that were used were the 5′RACEamplicon from start to 500 bp, the region from 450 bp to 1300 bp, andthe region from 1000 bp to the end of the transcript. The finalassembled sequence resulted in two versions of PiCAD.

Two regions, i.e. TBR2 and TBR3, were suggested being important fortoxin-receptor interaction by Gómez et al. (2003) BIOCHEMISTRY42(35):10482-9, Xie et al. (2005) J BIOL CHEM. 280(9):8416-25, and Chenet al. (2007) PROC NAT'L ACAD SCI. 104(35):13901-6. However, the clonedTnCAD and PiCAD sequences of the present invention are quite differentcompared to those from the other Lepidopteran insects. Based on thesequence alignment provided herein in FIGS. 1B and C, TBR3 modifications(as shown in FIG. 6C) were cloned into PiCAD. TBR3 (as shown in FIG. 1C)and TBR2 modifications (FIG. 1B) were cloned into TnCAD.

Example 4. Protein Expression

The receptor genes in the current disclosure were cloned intobaculovirus expression system (Gibco BRL Catalogue No. 10359-016)according to the manufacturer's provided protocols. Sequences wereverified using standard Sanger sequencing methods. Baculovirus stockswere created using Bac-to-Bac (Invitrogen) and BacMagic-3 kits from Lifetechnologies and Novagen, respectively.

Example 5. Demonstrate Binding of Bt Toxin and its Receptor

Ligand Blot Analysis

This example illustrates the binding of Cry toxin proteins to certain Tnor Pi CAD proteins or to modified Tn or Pi CAD proteins. Constructsexpressing TnCAD TBR3 variant and TBR2 variant were generated todetermine if the amino acid changes illustrated in FIGS. 1B and C resultin increased Cry1A-type toxin binding.

Ligand blotting of receptors with toxins is known in the art and wasused for demonstrating the specific binding of Cry toxins to bindingproteins/receptors. For non-limiting examples see: Xie, R., et al.(2005) J. BIOL. CHEM. 280: 8416-8425; Griko, N. B., et al. (2007)BIOCHEMISTRY 46:10001-10007.

Analysis of these two receptor polypeptides by ligand blot demonstratethat Cry1Ac (FIG. 2B) and Cry1Ab (FIG. 2A) tryptic cores bound to boththe MBP-fusion protein and the TVMV protease cleaved TnCAD TBR3 variant,whereas the wild type TnCAD protein did not bind to either toxin. Thetryptic cores of Cry proteins were produced by digesting the full-lengthCry protein with trypsin. Mutations in TnCAD TBR2 that had beenpreviously implicated in Cry1A toxin binding (Gomez et al. (2003)BIOCHEMISTRY. 2003 Sep. 9; 42(35):10482-9) had no effect.

Pulldown Analysis

Pulldown analysis were also performed to determine if TnCAD TBR2 variantand TBR3 variant interact with Cry1Ab tryptic core. Purified TnCAD TBR2or TBR3 was mixed with Cry1Ab or Cry1Ac. This mixture was thenimmobilized via C-terminal 6× Histidine tag of TnCAT TBR3 to NiNTAresin. The protein complex bound to the NiNTA resin was eluted andresolved by SDS-PAGE to determine if the bead-bound protein fractioncontained Cry1Ab tryptic core. The results of the pull-down experimentindicate that TnCAD TBR3 interacts with the tryptic core of Cry1Ab (FIG.3B) and that this region of Cadherin is involved in Cry1Ab toxinbinding.

Gel-Filtration

Another method to determine TnCAD TBR3 variant interaction with thetryptic core of Cry1Ab is gel filtration. Purified TnCAD TBR3 variantwas mixed with Cry1Ab tryptic core and purified through a HiLoad16/60Superdex200 gel filtration column. The peak fractions from gelfiltration were resolved by SDS-PAGE to check the purity and confirm theinteraction between TnCAD TBR3 variant and Cry1Ab. A distinct peaklabeled as Cadherin-Cry1Ab_TC in FIG. 3A was seen for the complex ofreceptor and toxin core before each individual protein labeled asCadherin or Cry1Ab_TC was detected (FIG. 3A).

Example 6. Determination of the Cadherin Ligand Binding Region

Two truncation variants of TnCAD TBR3 variant A (SEQ ID NO: 139), and B(SEQ ID NO: 140) (as shown in FIG. 4A) were designed to determine thetoxin binding region of Cadherin. The constructs were made withcleavable MBP fusion at the N-terminus and/or with tag N6His at theC-terminus or at the N-terminus. These constructs were expressed in E.coli, subsequently purified by Ni-NTA, and were then mixed with Cry1Abby molar ratio 1.2:1. This mixture was purified by a sizing column usingeither superdex75 or superdex200. Both proteins were confirmed to bindCry1Ab tryptic core as shown in FIGS. 4 B and C.

Example 7. Assess Binding Affinity of a Candidate Ligand to its Receptor

Assessing Binding Affinity of TnCAD TBR3 Variant (SEQ ID NO: 6) andToxin Candidates

Surface plasmon resonance (SPR) experiments were performed using aBiacore T000 instrument. Sensorgrams were processed with ScrubberVersion 2.0b, biologic Software (Campbell, Australia). To determine theaffinity of the TnCAD protein to toxins containing Cry1A domains, thepurified TnCAD and TBR3 variant TnCAD protein were immobilized on theBiacore chip. Toxins, including the tryptic cores (TC-) of TIC105,TIC107, and Cry1Ab were used as analytes and the affinity of eachprotein was determined towards TnCAD TBR3 variant. Binding wasdetermined for TIC107 and Cry1Ab (FIG. 3C) with respective Kd values of645 nM and 470 nM respectively. No binding was seen for TIC105 (FIG.3C).

Assessing Binding Characteristics of Sf ALP (SEQ ID NO:16) and ToxinCandidates

SPR experiments were also performed for SfALP and tryptic cores (TC_) ofCry2Ab and Cry1Ca. Binding kinetic of TC-Cry2Ab to SfALP is biphasic,indicating heterogeneity or two-phase/conformation change as shown inFIG. 5A. Binding of TC_Cry1Ca is significant as shown in FIG. 5B. WhileTC_Cry2Ab and TC_Cry1Ca showed binding to ALP, TC_Cry1Ac and TC_Tic105did not show interaction with ALP (FIG. 5C).

Example 8. Assessing Cytotoxicity of a Toxin

S. frugiperda (Sf9) cells obtained from ATCC (ATCC-CRL 1711) are grownat 27° C. in Sf-900 II serum free medium (Gibco BRL, Catalogue No.10902-088). These cells, which are not susceptible to some Bt toxins,are transfected with an expression construct for a Bt toxin receptordisclosed herein. Then the transfected Sf9 cells expressing the Bt toxinreceptor are exposed to one or more Bt toxins known to bind to the Bttoxin receptor and then stained using SYTOX Orange dye (used as anindicator of cell death) to detect compromised membranes. Bt toxins usedin this study include, but not limited to, Cry2Ab (GenBank accessionnumber: AAA22342), Cry1AC (GenBank accession number AA22331), TIC105described in U.S. Pat. No. 8,034,997 and TIC107 described in U.S. Pat.No. 7,741,118, all of which are incorporated herein by reference.

Cytotoxicity Assessment of PiCAD TBR3 Variant (as Set Forth in SEQ IDNO: 10)

The sequence changes to the variant modified PiCAD TBR3 are illustratedin FIG. 5C. PiCAD TBR3 variant and its wild type control were expressedin Sf9 cells and the transfected Sf9 cells were exposed various Bttoxins to determine if expression of TBR3 variant in cell line Sf9results in increased sensitivity to Cry1A-type toxins.

Cry1Ac and TIC107 (a Cry1A-type protein toxin) were used in bindingassays to qualitatively assess binding characteristics with wild typePiCAD. No effect was seen for the wild type PiCAD control demonstratingthat it is not sufficient in this context as a Cry1Ac or TIC107 receptor(FIG. 6A). However, expression of TBR3 variant resulted in increasedsensitivity to Cry1Ac and TIC107 (FIG. 6B). These results demonstratethat the modifications made by the present invention in the TBR3 regionsequence are responsible for Bt toxin receptor binding and responsiblefor driving receptor oligomerization and pore formation of the toxinresulting in cell death.

Cytotoxicity Assessment of Spodoptera frugiperda ABC Transporter (as SetForth in SEQ ID NO:15)

Spodoptera frugiperda ABC transporter was expressed by baculovirusmediated infection of SF9 insect cells. Briefly, 50,000 SF9 cells wereseeded two days prior on poly-D lysine coated plates. After cellattachment, the cells were infected with a 1:50 dilution of a P3 viralstock for expression of ABC transporter. The cells were challenged with50 ppm of Cry1Ac, TIC107 or buffer control (50 mM CAPS pH 10.8, 10 mMDTT) after 42 hours. A Safire 2 plate reader (488 excitation/530emission) was used to record values on the cells after 2 hours.Representative images as shown in FIG. 7A were captured on an invertedmicroscope equipped with a GFP filter as well as a bright field image ofthe same area.

Table 4 shows a number of ABC transporters identified from thecytotoxicity assessment as Bt toxin receptors by the presentapplication.

TABLE 4 PRT Sytox Green Fluresence SEQ ID control receptor NO toxintreatment buffer HzABCa7 172 Cry2AB 55345 5398 SfABCa3 177 Cry2AB 306485398 SfABCc2 181 Tic105 14928 645 SfABCc2 181 Cry1Ac 20686 645 SfABCc337 Tic105 17667 645 SfABCc3 37 Cry1Ac 18560 645 HzABCc2 185 Tic105 387801104 SfABCc5 183 Tic107 33024 5398

Example 9. Differentiate Toxin MOA Via dsRNA Suppression of ToxinReceptors

Double-stranded RNAs (dsRNA) as set forth in SEQ ID NO: 147 and 148corresponding to Cry3Bb receptors/binding partners, e.g., WCR Cadherin(seq ID NO:55) and ADAM metalloprotease (SEQ ID NO: 20) respectivelywere fed to Diabrotica virgifera virgifera (also referred as WesternCorn Rootworm) neonate larvae for 4 days to knock-down the candidatereceptor gene. The larvae were then transferred to a new diet platecontaining the toxin (2000 to 6000 ppm) for 6 days. The controls arebuffer and dsRNA-only samples to assure dsRNA by itself does not causeinsect mortality.

Larvae from each sample were measured for the percentage of an 8-insectpopulation that exhibited mortality or 2nd/3rd-instar stunting on thetenth day. This percentage is also termed percent effective control (%EC). Replicates of 8-insect populations were averaged for a mean % EC(±SE on the mean).

Decreased Cry3Bb toxicity indicates that the candidate gene encodes aprotein involved in Cry3Bb toxicity. This protein can be a receptor, abinding partner or a protein involved in secondary events to the initialbinding interaction. In addition, larval samples at day 4 and day 11were submitted to RNA extraction and real-time PCR to verify genetranscript knock-down.

The results showed that the dsRNA targeting Cadherin or ADAMmetalloprotease conferred Cry3Bb resistance (FIG. 8), and the combineddsRNA targeting both Cadherin and ADAM metalloprotease simultaneouslyconferred synergistic Cry3Bb resistance. None of these dsRNA moleculeshad any statistically significant effect on TIC1201 toxicity in WesternCorn Rootworm. These results demonstrated that cadherin and ADAMmetalloprotese are essential for Cry3Bb toxicity, but not for TIC1201toxicity, indicating the differences in MOA by Cry3Bb and TIC1201.Therefore combining or stacking Cry3Bb and TIC1201 will be effective inreducing the risk of resistance when mutations in Cadherin or ADAMmetalloprotease are potential underlying mechanisms (Morin, Shai et al.Proc Natl Acad Sci USA. 2003 Apr. 29; 100(9): 5004-5009).

Example 10. Identify Bt Toxin Receptors from Diabrotica virgiferavirgifera

cDNA libraries were generated from mid-guts of Diabrotica virgiferavirgifera (Western corn rootworm, WCR) third instar larvae reared oncorn plants and sequenced by high-throughput sequencing usingcommercially available 454 technology (454 Life Sciences, 15 CommercialSt., Branford, Conn. 06405, USA), as described in Margulies et al.(2005) NATURE, 437:376-380. This provided approximately 1.27 million˜300 base-pair reads, which were supplemented with 17,800 publiclyavailable ˜520 base-pair Sanger reads from NCBI. The combined sequencedata were assembled into contigs de novo using the Newbler (version 2.3)software package (454 Life Sciences, 15 Commercial St., Branford, Conn.06405, USA). Approximately 16,130 genes were identified from theassembled sequence data.

For sequence annotation, Blast based annotation was performed by usingNCBI's Blastall 2.2.21 software to search Diabrotica virgifera virgiferacontigs against the publicly available uniref90.fasta database(ftp.uniprotorg/pub/databases/uniprot/current_release/uniref/uniref90/).The blast search was performed in blastx mode (translated Diabroticavirgifera virgifera nucleotide queries searched against the uniref90protein database). Only blast hits with an e-value less than or equal to9e-9 were retained. For each Diabrotica virgifera virgifera contig thedescription line from the uniref90 best hit was used as an annotation.When no Blast hits were found, the sequence was subjected to asupplementary Pfam search. To accomplish this, the longest open readingframe (ORF) was identified for each Diabrotica virgifera virgiferacontig and used to query the publicly available Pfam-A database(ftp.sanger.ac.uk/pub/databases/Pfam/current_release) using the publiclyavailable HMMER 3.0 software package (hmmer janelia.org/). Diabroticavirgifera virgifera contigs with a Pfam hit with an e-value less than orequal to 1e-5 were annotated with the protein family name and the Pfamidentifier.

TABLE 5 Bt toxin receptors identified from Diabrotica virgiferavirgifera NUC PEP SEQ SEQ ID ID Gene identifier NO NO annotationDiabrotica_virgifera_virgifera_ 45 92 ABC transporterABC_transporter_105_10 Diabrotica_virgifera_virgifera_ 46 93 ABCtransporter ABC_transporter_105_9 Diabrotica_virgifera_virgifera_ 47 94ABC transporter ABC_transporter_218_1 Diabrotica_virgifera_virgifera_ 4895 ABC transporter ABC_transporter_218_2 Diabrotica_virgifera_virgifera_49 96 ABC transporter ABC_transporter_218_3Diabrotica_virgifera_virgifera_ 50 97 ABC transporterABC_transporter_218_4 Diabrotica_virgifera_virgifera_ 51 98Aminopeptidase Aminopeptidase_837_1 Diabrotica_virgifera_virgifera_ 5299 Aminopeptidase Aminopeptidase_837_2 Diabrotica_virgifera_virgifera_53 100 Aminopeptidase Aminopeptidase_871_1Diabrotica_virgifera_virgifera_ 54 101 cadherin like cadherin_1083_1protein Diabrotica_virgifera_virgifera_ 55 102 cadherin likecadherin_1817_1 protein Diabrotica_virgifera_virgifera_ 56 103 ABCtransporter ABC_transporter_01859_1 Diabrotica_virgifera_virgifera_ 57104 ABC transporter ABC_transporter_01867_1Diabrotica_virgifera_virgifera_ 58 105 ABC transporterABC_transporter_01873_1 Diabrotica_virgifera_virgifera_ 59 106Aminopeptidase Aminopeptidase_01949_1 Diabrotica_virgifera_virgifera_ 60107 ADAM ADAM_metalloprotease_01952_1 metalloproteaseDiabrotica_virgifera_virgifera_ 61 108 AminopeptidaseAminopeptidase_02024_1 Diabrotica_virgifera_virgifera_ 62 109Aminopeptidase Aminopeptidase_02031_1 Diabrotica_virgifera_virgifera_ 63110 Aminopeptidase Aminopeptidase_02119_1Diabrotica_virgifera_virgifera_ 64 111 ADAM ADAM_metalloprotease_02122_1metalloprotease Diabrotica_virgifera_virgifera_ 65 112 AminopeptidaseAminopeptidase_02140_1 DIADiabrotica_virgifera_virgifera_ 66 113Aminopeptidase Aminopeptidase_02340_1 Diabrotica_virgifera_virgifera_ 67114 ABC transporter ABC_transporter_02470_1Diabrotica_virgifera_virgifera_ 68 115 ABC transporterABC_transporter_02630_1 Diabrotica_virgifera_virgifera_ 69 116 AlkalineALP_02713_1 phosphatase Diabrotica_virgifera_virgifera_ 70 117Aminopeptidase Aminopeptidase_03898_1 Diabrotica_virgifera_virgifera_ 71118 ADAM ADAM_metalloprotease_04620_1 metalloproteaseDiabrotica_virgifera_virgifera_ 72 119 ADAM ADAM_metalloprotease_04627_1metalloprotease Diabrotica_virgifera_virgifera_ 73 120 AminopeptidaseAminopeptidase_04697_1 Diabrotica_virgifera_virgifera_ 74 121Aminopeptidase Aminopeptidase_04881_1 Diabrotica_virgifera_virgifera_ 75122 cadherin cadherin_04907_1 Diabrotica_virgifera_virgifera_ 76 123Aminopeptidase Aminopeptidase_05042_1 Diabrotica_virgifera_virgifera_ 77124 ADAM ADAM_metalloprotease_05390_1 metalloproteaseDiabrotica_virgifera_virgifera_ 78 125 ABC transporterABC_transporter_05581_1 Diabrotica_virgifera_virgifera_ 79 126 ADAMADAM_metalloprotease_05844_1 metalloproteaseDiabrotica_virgifera_virgifera_ 80 127 ABC transporterABC_transporter_06637_1 Diabrotica_virgifera_virgifera_ 81 128 ADAMADAM_metalloprotease_07383_1 metalloproteaseDiabrotica_virgifera_virgifera_ 82 129 ABC transporterABC_transporter_07661_1 Diabrotica_virgifera_virgifera_ 83 130 ADAMADAM_metalloprotease_08650_1 metalloproteaseDiabrotica_virgifera_virgifera_ 84 131 AminopeptidaseAminopeptidase_08778_1 Diabrotica_virgifera_virgifera_ 85 132Aminopeptidase Aminopeptidase_08810_1 Diabrotica_virgifera_virgifera_ 86133 Aminopeptidase Aminopeptidase_09768_1Diabrotica_virgifera_virgifera_ 87 134 cadherin like cadherin_10167_1protein Diabrotica_virgifera_virgifera_ 88 135 ADAMADAM_metalloprotease_10594_1 metalloproteaseDiabrotica_virgifera_virgifera_ 89 136 ABC transporterABC_transporter_11225_12 Diabrotica_virgifera_virgifera_ 90 137 ABCtransporter ABC_transporter_11255_1

Example 11. Microarray Methods

Nucleic acid molecules of the present invention can be used to monitorexpression of target sequences. A microarray-based method forhigh-throughput monitoring of gene expression may be utilized to measuregene-specific hybridization targets. This ‘chip’-based approach involvesusing microarrays of nucleic acid molecules as gene-specifichybridization targets to quantitatively measure expression of thecorresponding genes (Schena et al., SCIENCE 270:467-470 (1995). Everynucleotide in a large sequence can be queried at the same time.Hybridization can be used to efficiently analyze nucleotide sequences.Several microarray methods have been described in the literature. Onemethod compares the sequences to be analyzed by hybridization to a setof oligonucleotides or cDNA molecules representing all possiblesubsequences (Bains and Smith, J. THEOR. BIOL. 135:303 (1989)). A secondmethod hybridizes the sample to an array of oligonucleotide or cDNAprobes. An array consisting of oligonucleotides or cDNA moleculescomplementary to subsequences of a target sequence can be used todetermine the identity of a target sequence, measure its amount, anddetect differences between the target and a reference sequence. Nucleicacid molecule microarrays may also be screened with protein molecules orfragments thereof to identify nucleic acid molecules that specificallybind protein molecules or fragments thereof. The microarray approach mayalso be used with polypeptide targets of the current invention.Essentially, polypeptides are synthesized on a substrate (microarray)and these polypeptides can be screened either with protein molecules orfragments thereof or nucleic acid molecules in order to screen forprotein molecules or fragments thereof or for nucleic acid molecules

What is claimed is:
 1. A recombinant DNA molecule encoding a polypeptidederived from Spodoptera frugiperda having an amino acid sequenceselected from the group consisting of: a) an amino acid sequence as setforth in SEQ ID NO: 181; b) an amino acid sequence having at least 90%sequence identity to the full length of the amino acid sequence setforth in SEQ ID NO: 181; and c) an amino acid sequence having at least95% sequence identity to the full length of the amino acid sequence setforth in SEQ ID NO: 181; wherein said recombinant DNA molecule isoperably linked to a heterologous promoter capable of initiating thetranscription of the DNA sequence, and wherein said polypeptide has Bttoxin binding activity.
 2. The recombinant DNA of claim 1, wherein saidrecombinant DNA molecule has a nucleotide sequence of SEQ ID NO:
 162. 3.A recombinant DNA vector comprising the recombinant DNA moleculeaccording to claim
 1. 4. A non-human transgenic cell transformed withthe recombinant DNA molecule according to claim
 1. 5. The transgeniccell of claim 4, wherein said transgenic cell is an insect cell.
 6. Anon-human transgenic organism transformed with the recombinant DNAmolecule according to claim
 1. 7. The transgenic organism according toclaim 6, wherein said transgenic organism is a prokaryotic or aeukaryotic organism.
 8. The transgenic organism of claim 6, wherein saidtransgenic organism is a whole insect.
 9. A method to assess thetoxicity of a candidate ligand, wherein said method comprises the stepsof: a) contacting said candidate ligand with cells comprising therecombinant DNA molecule of claim 1 and expressing said polypeptide withBt toxin binding activity; and, b) measuring the toxicity effect of saidcandidate ligand on said cells in terms of cell death indices.